The present invention relates to a device and a method for performing a biological modification of a fluid, and more particularly to a device and method for assisting or replacing an organ which normally performs such a modification of the fluid.
A number of organs in the body, such as the liver, modify fluids such as blood. The liver is a particularly complex organ because it acts both as a filter and as an active metabolic unit. As a filter, the liver removes toxic substances from the blood. In addition, the liver performs many biochemical functions such as detoxifying ammonia into urea, bilirubin metabolism, glycogen storage, lipid synthesis, drug metabolism, albumin secretion and clotting factor secretion. Thus, the liver has many important functions within the body which render it essential. If the liver should fail, the body would be unable to continue operating.
There are many causes of liver failure, including exposure to toxic substances, hepatitis, and genetic defects (Kasai, et. al. . Artif. Organs, 18:348-54, 1994). Currently, 70% of patients with acute liver failure die because of no available treatment (Kasai, et. al. . Artif. Organs, 18:348-54, 1994). Furthermore, 10-30% of patients die while awaiting donor liver organs (LePage, et al., Am. J Crit. Care, 3:224-7, 1994; Sussman, et al. Artif. Organs, 18:390-6, 1994; and Uchino and Matsushita, Asaio J., 40:74-7, 1994).
A bedside life-support device that could temporarily perform liver function during liver failure is called an Extracorporeal Liver Assist Device (ELAD). The development and commercialization of such a device would clearly be of enormous benefit for a number of reasons (Fox et al, Am. J. Gastroenterol., 88:1876-81, 1993). An ELAD would benefit the roughly 2,000 patients with fulminant liver failure (FH) in the U.S. each year (Hoofnagle, et al., Hepatology, 21:240-52, 1995). It could also be used as a bridge to liver transplantation for patients awaiting donor organs.
An ELAD that would function for several weeks could in addition allow for recovery to normal functioning of the patient""s own liver. Since it is unlikely that every hepatocyte is destroyed in a damaged liver, adequate liver support for two to three weeks could allow surviving hepatocytes to repopulate the damaged liver. Fewer than a dozen hepatocytes are required to repopulate the liver in an animal model of lethal hepatic disease (Sandgren et al., Cell, 66:245-56, 1991). A patient with 90-95% liver necrosis should be able to recover sufficient function to survive independently after only a few days of support (Sussman et al. Artif. Organs, 18:390-6, 1994).
In an attempt to provide such an ELAD, several purely mechanical, non-biological blood-treatment devices have been developed. In the most basic form, the purpose of these devices is to selectively remove toxins and add nutrients across a membrane with a relatively small pore size. One of the most advanced of these non-biological devices has been developed by Hemocleanse(trademark) and has recently received FDA approval. In a randomized, controlled clinical trial using the Hemocleanse(trademark) apparatus, removal of metabolites was limited and there was no significant effect on blood ammonia levels (Hughes et al., Int. J. Artif. Org., 17:657-662, 1994). Clearly, liver function is extremely complex and is unlikely to be replaced by a solely mechanical or a chemical device at this time.
Other currently available ELADs use biological materials as a starting point. For example, one of the most clinically tested ELADs uses a transformed immortalized human cell line as a source for hepatocyte-like cells (Sussman, et al., Artif. Organs, 18:390-6, 1994). Initial trials of this device were performed under xe2x80x9cEmergency Use of Unapproved Medical Devicesxe2x80x9d, or xe2x80x9cInvestigational Device Exemptionxe2x80x9d. Efficacy was not determined, but no serious adverse side effects were observed except for clotting that was managed by drug treatment. While the use of an immortalized human cell line is convenient because it provides an expendable source of cells, there are two major reasons why it may not be ideal. Firstly, there are obvious safety and regulatory concerns about using immortalized cell lines in clinical practice. Secondly, immortalized cells would not be expected to retain all the normal physiological characteristics of primary hepatocytes, particularly after industrial scale expansion (Sussman et. al., Artif Organs, 18:390-6, 1994).
A second general approach for obtaining liver cells as a source for an ELAD, is the isolation of liver cells or tissue from intact livers. In previous attempts, cells from livers have usually been disassociated using enzymes such as collagenase, which disrupts the normal micro architecture of the liver. Some attempts have been used to use liver pieces, but the shape of these pieces have not been designed for proper surface area to volume ratios necessary for optimal tissue maintenance (Lie et al., Res Exp Med (Berl) 185:483-94, 1985)
One current limitation is the ability of current methods of culturing mammalian liver cells to provide conditions which allow cells to assemble into tissues which simulate the spatial three-dimensional form of actual tissues in the intact organism. Conventional tissue culture processes limit, for similar reasons, the capacity for cultured tissues to express a highly functionally specialized or differentiated state considered crucial for mammalian cell differentiation and secretion of specialized biologically active molecules of research and pharmaceutical interest. Unlike microorganisms, the cells of higher organisms such as mammals form themselves into high order multicellular tissues. Although the exact mechanisms of this self-assembly are not known, in the cases that have been studied thus far, development of cells into tissues has been found to be dependent on orientation of the cells with respect to each other or another anchorage substrate and/or the presence or absence of certain substances such as hormones. In summary, no conventional culture process used in the organ assist devices to date is capable of simultaneously achieving proper functioning of the cells in vitro while at the same time maintaining critical cell/cell/substrate interactions and proper microenvironment to allow excellent modeling of in vivo organ tissue structure and function.
In the liver, the unique juxtaposition of diverse cell populations and matrix components in harmony with the angio architecture results in a delicate bioecological system. It is therefore unlikely that standard cell cultures of hepatocytes will perform even the minimal liver functions. As mentioned previously, the cells of higher organisms such as mammals form themselves into high order multicellular tissues. An example of physical contact between a cell and a noncellular substrate (matrix) is the physical contact between an epithelial cell and its basal lamina. Examples of functional contact between one cell and another cell includes electrical or chemical communication between cells. For example, cardiomyocytes communicate with other cardiomyocytes via electrical impulses. In addition, many cells communicate with other cells via chemical messages, e.g., hormones, which either diffuse locally (paracrine signaling and autocrine signaling), or are transported by the vascular system to more remote locations (endocrine signaling). Examples of paracrine signaling between cells are the messages produced by various cells (known as enteroendocrine cells) of the digestive tract, e.g., pyloric D cells which secrete somatostatin which in turn inhibits the release of gastrin by nearby pyloric gastrin (G) cells.
This microarchitecture can be extremely important for the maintenance of a tissue explant of an organ in minimal media, e.g., without exogenous sources of serum or growth factors, because the liver tissue can be sustained in such minimal media by paracrine and autocrine factors resulting from specific cellular interactions within the micro-organ,
The preparation of such a micro-organ culture is described in U.S. patent application Ser. No. 08/482,364, herein incorporated by reference. In the preparation of a micro-organ culture, the populations of cells which make up the explant are isolated from a liver in a manner that preserves the natural affinity of one cell to another, e.g., to preserve layers of different cells as present in the organ itself For example, in skin micro-organ cultures, keratinocytes of the epidermis remain associated with the stroma and the normal tissue architecture is preserved including the hair follicles and glands. This basic structure is common to all organs, for instance, which contain an epithelial component. Moreover, such an association facilitates intercellular communication. This is particularly important in differentiating cells where induction is defined as the interaction between one (inducing) and another (responding) tissue or cell, as a result of which the responding cells undergo a change in the direction of differentiation. Moreover, inductive interactions occur in embryonic and adult cells and can act to establish and maintain morphogenetic patterns as well as induce differentiation (Gurdon, Cell, 68:185-199, 1992).
Furthermore, the micro-organ cultures prepared according to U.S. patent application Ser. No. 08/482,364 preserve normal liver tissue architecture even when cultured for prolonged periods of time. Because these cultures can be maintained in controlled and uniform conditions and yet closely resemble tissue in vivo, they provide a unique continuous source of functional liver cells in vitro.
Unfortunately, none of the prior art organ assist devices, or related devices in the prior art, uses micro-organ cultures to perform a biological modification of a fluid.
Therefore, there is a decided need in the art for a device and a method for performing a biological modification of a fluid, particularly for assisting or replacing a failed organ of a subject, which can perform the functions of the organ and which includes a micro-organ culture.
According to the present invention there is provided a device for performing a biological modification of a fluid, the device comprising: (a) a chamber having an inlet for intake of the fluid and an outlet for outflow of the fluid, and (b) a collection of micro-organ cultures of an organ for performing the biological modification of the fluid, each individual micro-organ culture of the collection including cells and having dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture (organ structure) of organ units (e.g., acinus units of liver) is maintained within each individual micro-organ culture, the collection of micro-organ cultures being located within the chamber and the collection of micro-organ cultures being in contact with at least a portion of the fluid flowing through the chamber.
As used herein, the term xe2x80x9cMCxe2x80x9d refers to micro-organ culture.
Preferably, the organ is liver. Also preferably, the collection of micro-organ cultures includes cells from the organ, such that intercellular contacts between the cells are preserved. Most preferably, each of the collection of micro-organ cultures is characterized by an Aleph of at least about 2.6 mmxe2x88x921.
According to preferred embodiments of the present invention, the micro-organ culture is substantially encapsulated by a sheet of a biocompatible polymer, the sheet being located substantially within the chamber. Preferably, the sheet has a first dimension in a range of from about 30 cm to about 90 cm, a second dimension in a range of from about 30 cm to about 80 cm and a third dimension in a range of from about 300 micrometers to about 900 micrometers. Also preferably, a plurality of the sheets are incorporated substantially parallel in orientation within the chamber, such that fluid flows freely between the sheets.
According to another embodiment of the present invention, there is provided a device for performing a biological modification of a fluid of a subject, including: (a) a chamber having an inlet for intake of the fluid and an outlet for outflow of the fluid; (b) a collection of micro-organ cultures for performing the biological modification of the fluid, each individual micro-organ culture of the collection including cells and having dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture of organ units is maintained within each individual micro-organ culture, the collection of micro-organ cultures being located within the chamber and the collection of micro-organ cultures being in contact with at least a portion of the fluid flowing through the chamber; (c) a first tube having first and second ends, the first end for coupling to the subject for receiving the fluid from the subject, the second end for coupling to the inlet; and (d) a second tube having first and second ends, the first end for coupling to the outlet and the second end for coupling to the subject to return the fluid to the subject after the biological modification.
According to still further embodiments of the present invention, there is provided a method of performing a biological modification of a fluid from a subject, the method comprising the step of perfusing a chamber containing a collection of micro-organ cultures with the fluid from the subject, such that the collection of micro-organ cultures performs the biological modification on the fluid, wherein each individual micro-organ culture of the collection includes cells and has dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 150 micrometers and not more than about 225 micrometers away firm a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture of organ units is maintained within each individual micro-organ culture.
According to still further embodiments of the present invention, there is provided a method of preparing a continuous planar organ. The method comprising the steps of (a) obtaining a collection of individual micro-organ cultures of an organ, such that each of the individual micro-organ culture of the collection includes cells and has dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture of organ units is maintained within each individual micro-organ culture; and (b) adding (e.g., layering) a suspension of cells from the organ onto the micro-organ cultures and coculturing the suspension of cells in presence of the collection of micro-organ cultures, such that the continuous planar organ is formed from an admixture of cells derived from the micro-organ cultures and the cells in suspension.
According to a preferred embodiment of the present invention, the collection of liver micro-organ cultures is provided within a continuous liver planar organ formed by culturing hepatocyte cells in presence of the collection of liver micro-organ cultures, such that the continuous liver planar organ is formed from an admixture of cells derived from the micro-organ cultures and the hepatocyte cells.
According to still further embodiments of the present invention, there is provided a method of preparing a continuous liver planar organ. The method comprising the steps of (a) obtaining a collection of individual liver micro-organ cultures, such that each of the individual micro-organ culture of the collection includes liver cells and has dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo liver architecture of acinus units is maintained within each individual micro-organ culture; and (b) adding (e.g., layering) a suspension of hepatocyte cells onto the micro-organ cultures and coculturing the suspension of cells in presence of the collection of liver micro-organ cultures, such that the continuous planar liver organ is formed from an admixture of cells derived from the micro-organ cultures and the hepatocyte cells.
Additional features of the invention are described hereinunder.